Kaposi's sarcoma-associated herpesvirus (KSHV), a prominent human pathogen accounting for a large percentage of virus-induced cancers worldwide, has evolved a variety of stratagems for evading host immune responses to establish life-long persistent infection. Early detection of invading viruses by the host depends on a limite number of specific intracellular sensors that can detect viruses' conserved patterns and activate type I interferon (IFN)-mediated anti-viral mechanisms. The cGAS acts as a cytosolic pathogen DNA sensor and subsequently generates the second messenger cGAMP that binds the downstream molecule STING, leading to IFN production against herpesviral infection. Furthermore, TRIM56 physically links cGAS and STING to generate a novel linear cGAS-TRIM56-STING signaling pathway. On the other hand, the KSHV ORF32 tegument protein specifically targets TRIM56 and blocks cGAS-TRIM56-STING-mediated IFN signaling pathway. In addition, we have developed not only a new infectious bacterial artificial clone (BAC) of the KSHV genome for genetic manipulation and infectious virus production, but also a NOD/SCID IL2R-/- (NSG) humanized mouse model for in vivo KSHV persistence. A main hypothesis is that the linear cGAS-TRIM56- STING DNA sensing pathway plays a critical role in host anti-herpesviral immunity and that KSHV has evolved to carry the ORF32 tegument protein to disable this host DNA sensing pathway to facilitate persistent infection. In this proposal, we will utilize multidisciplinary approaches to define the host's linear cGAS-TRIM56-STING pathway through the use of CRISPR genome editing, single-molecule technique, and knockout cells and mice (Aim 1). We will delineate the evasion mechanisms of KSHV ORF32 and other herpesviral homologs using BAC mutagenesis and in vitro infection (Aim 2). Finally, we will test whether loss of the ORF32-mediated inhibition of cGAS-TRIM56-STING pathway affects KSHV persistence in NSG humanized mice (Aim 3). This proposal is highly innovative and its successful outcome should significantly enhance our understanding of host DNA sensing-mediated innate immunity and viral evasion, ultimately contributing to the fields of AIDS- associated malignancy as well as opportunistic infections.